"Emir KHATIPOV" <khatipov at nibh.go.jp> wrote:
Next time you have a need for information, please don't splatter your
question across a wide, diverse range of Usenet groups and ask for
the responses in email. Followups set to sci.chem.analytical.
>Does one have to follow the same precautions when attaching/detaching HPLC
>columns, as in the case of low pressure column chromatography, to avoid air
>bubble penetration into the column?
HPLC columns typical operate from 500-5000 psi, hence the small effect
of gravity compared to the liquid head of packed gravity columns.
Because of the small particle size in HPLC columns, the fluid is held
by forces such as capillary action, consequently the orientation
of most analytical columns is not important and gravity will not
drain mobile phase from the column. Columns are usually
stored in relatively non-volatile mobile phases to prevent air
entrainment on storage - however the solvent will slowly evaporate
if the ends are not well-plugged, thus the columns should not be
stored with buffers - unless specifically recommended by manufacturers.
Most long-term stored columns will lose solvent ( unless using solvent
bellows or similar devices ), however normal procedures will ensure
the column is carefully filled during startup.
It is possible to disrupt the bed of sensitive HPLC columns by
changing solvents too quickly, or applying pressure too quickly.
It's also important to ensure storage solvents ( pump, injector,
column, and detector ) are all miscible with the mobile phase.
If necessary an intermediate solvent, is used to ensure all
solvents remain miscible during flushing.
Most people check the pump flow rate at the nominal flow when
starting, then stop the flow and connect the column. It's usual
check the column pressure and flow relationship before connecting
the detector.
Most column/pump manuals provide suggesions on the rate of flow increase
for columns during starting - especially for columns that are very
pressure sensitive or that contain material that swells or shrinks
with different solvents. The normal method is to slowly increase
the flow from the pump over a few minutes whilst watching and
measuring the column outlet flowrate to ensure the outlet flow
matches pump flow ( indicating no leaks ). The gradual increase
in flow will also ensure no vapour or air voids are present
( regardless of column orientation ).
In general, great care should be exercised to ensure solvents used
in gradients or increased column temperatures are thoroughly
degassed. As water content decreases, or column temperature
increases, gas solubility usually decreases, so the pressure
drop after passing through the column can cause disruption if
mobile phase flow is suddenly stopped, or the column
disconnected before the pressure has dropped.
HPLC columns are tending to get smaller ( 3 - 3.5um particles,
and column lengths of 100-150mm ) and may have less of the
separating material present - thus it's more important to be
concerned about mobile-phase quality and sample clean-up, as
good startup and shutdown procedures are already used to prolong
column life. I suspect details of precautions are available
from most column and equipment suppliers, and there are
several good HPLC texts available, including
Practical HPLC Method Development - 2nd edition
L.R.Snyder, J.J.Kirkland, and J.L.Glajch.
John Wiley & Sons (1997) ISBN 0-471-00703-X
Bruce Hamilton